It is a goal in this art to detect various nucleic acid sequences in a biological sample, in which the said sequences, as so-called target nucleic acid, is present in small amounts relative to its existence amongst a wide variety of other nucleic acid species including RNA, DNA or both. Thus, it is desirable to detect the nucleic acid encoding polypeptides that may be associated with pathological diseases or conditions, such as, for example, DNA correlating to that of the human immunodeficiency virus. In addition to the detection of nucleic acids encoding such viral particles, it is desirable to detect other nucleic acids characteristic of a pathological disease or condition such as a defective gene, as in the case of hemophilia, or in the detection of anti-pathogen antibodies of such diseases or conditions.
Characteristically, the nucleic acids associated with such are present, if at all, in very small amounts relative to total nucleic acid in a given biological sample, such as blood or other body fluid or tissue sample of a given individual to be tested.
Other important cases where the application of such technology finds use are detailed in said U.S. Ser. No. 852,692 and need not be repeated here.
The detection of such nucleic acid species requires such specificity that, if present, it is detectable and measurable from amongst the wide variety of other nucleic acid species with which it is environmentally associated. Some of these species may bear close homology, at least in isolated segments, with the target nucleic acid. Further, as noted above, these target nucleic acid species are very often found only in very minute amounts in the biological sample being tested. And yet, for proper diagnosis of the underlying disease state, it is essential that even small amounts of such target nucleic acid be detectable unequivocably for fidelity of the assay system.
Two fundamental approaches have been advanced for accomplishing the goal of the art. In one, the amount of nucleic acid in the sample is not altered or affected. Instead, a reporter system is developed whereby a large number of detectable molecules corresponding to the nucleic acid target are produced for ready detectability and measurement. Such a reporter system is a signal-generating system associated with the target nucleic acid producing a detectable signal representative of the number of molecules of target sequence. Such systems have employed a chromophore generating moiety linked to a oligonucleotide probe that hybridizes with the target nucleic acid sequence. The chromophore moiety can be isolated from those oligonucleotide probes that have properly hybridized to target, and measured. One such chromophore generating group is an enzyme such as alkaline phosphatase which has a chromogenic substrate producing under suitable conditions detectable and measurable colored molecules. Another such system employs radioactive labeling of the nucleic acid probe such that the signal generated by such properly hybridized target nucleic acid can be detected and measured.
A second approach has been developed that is fundamentally different in that it involves increasing the copy number of the target nucleic acid sequence itself, in particular in an amount greater than that of nucleic acid sequences with which it is associated in the sample. This can be done by selective amplification of the target nucleic acid sequence. One can refine the culture techniques of the sample such that somehow the target nucleic acid sequence is amplified preferentially to other nucleic acid sequences. These techniques are cumbersome and time consuming and subject to trial and error.
Another example of the second approach is amplification of a target nucleic acid sequence in a so-called "polymerase chain reaction" (PCR). This technique was reported by Saiki et al., Science 230, 1350 (1985) and Mullis et al., European Patent Application Publication Nos. 200362 and 201184 (See also U.S. Pat. Nos. 4,683,195 and 4,683,202), and particularly entails (1) hybridizing to a segment of target nucleic acid sequence a primer, (2) extending said primer with a polymerase, and (3) rendering single stranded the duplexes resulting from the chain extension reaction. This procedure can be repeated over a number of cycles so as to amplify the underlying target nucleic acid sequence. The procedure requires at least two nucleic acid probes and has three steps for a single cycle.
Certain RNAs are known to be susceptible to replication by certain polymerases, such as bacterial phage RNA-dependent RNA polymerase such as Q.beta. replicase and the replicase from brome mosaic virus (BMV). In this technique, the RNA can serve as a sequence template for replication by the RNA polymerase resulting in an amount of replicated RNA sequences that is an exponential increase of the amount initially present. See Miele et al., J. Molecular Biology 171, 281 (1983). A system in which probe for a target sequence is linked to an RNA capable of being replicated by Q.beta. replicase is described by Chu et al., Nucleic Acids Research 14, 5591 (1986) and by BMV replicase by March et al., Positive Strand RNA Viruses, Alan R. Liss (Publisher; New York) (1987; Proceedings of UCLA Symposium, 1986).
Until recently it has not been appreciated that (autocatalytic) replication could be employed to provide convenient, broadly applicable, highly sensitive reporter systems for analyses of nucleic acid sequences. Above-cited U.S. Ser. No. 852,692 provides the use of nucleic acid probe-replicative RNA adducts for use in detecting target nucleic acid sequences by amplification thereof via the exponential replicative process of the replicative RNA associated with the nucleotide probe. Thus, that invention combines the art of replication of RNA with the use of oligonucleotide hybridization probes to detect target nucleic acid by associated replicative amplification. Details of that invention can be readily adduced by reference to the co-pending patent application or its counterpart, published international application, both cited supra. One practical drawback of that method resides in its necessary use of relatively long, hence sensitive, sequences of replicatable RNA that may prove inherently unstable in the assay environment.
It is an object of the present invention to take further advantage of the basic replicative process for amplification, for ease in the detection of sequences corresponding to target nucleic acid sequences. It is a further object of the present invention to take advantage of other biological processes that serve in result to achieve amplification of a given nucleic acid sequence. In particular, advantage is taken of the natural transcription process (as the first step in expression of DNA to produce polypeptide products) whereby double-stranded nucleic acid templates containing a promoter sequence recognized by a DNA-dependent RNA polymerase is used to produce a plurality of corresponding RNA transcripts. Again, using this process, a large number of RNA transcripts can be produced,. that are themselves replicatable.
It is a further object of the present invention to combine the advantages of the replicative and transcript-producing procedures as a means for detecting and measuring corresponding target nucleic acid.
It is thus an object of the present invention to produce, in all events, a given RNA transcript sequence that corresponds by presence and amount to target nucleic acid sequence and that can be replicated to a plurality and that can be adapted by association with a signal grouping that is accountable for its detection and measurement.
It is thus an overall object of the present invention to meet the goals enumerated by the art and to overcome the disadvantages and problems encountered by prior researchers' endeavors. The present invention utilizes, if at all, only relatively short, hence stable, RNA sequences that need only contain a sequence that insures replicatability and nothing more. Thus, the present invention provides a straightforward technique that can be utilized with stable fidelity in an acceptably short period of time, employing the convenience of known reagents and having the precision necessary to reach consistent scientific results; one that can be employed in a reproducible assay setting and that is adaptable for use in kits for laboratory/clinical analyses. It is, hence, an object of the present invention to increase the detectability of certain nucleic acid sequences (target segments) by amplification of sequences associated with the presence of the target sequences in an in vitro or ex vivo system, utilizing the advantages provided by the natural transcription and replicative processes per se.